Summary:[unreadable] [unreadable] Burkholderia pseudomallei, the etiological agent of melioidosis, is a Gram-negative, facultatively anaerobic, motile bacillus that is responsible for a broad spectrum of illnesses observed in both humans and animals. Burkholderia mallei, the etiological agent of glanders, is a Gram-negative bacterium that is responsible for disease in donkeys, mules, horses and occasionally humans. Unlike the environmental saprophyte B. pseudomallei, however, B. mallei does not persist in nature outside of its soliped hosts. While B. mallei and B. pseudomallei are genotypically similar, significant phenotypic differences do exist between the two pathogenic species. Although glanders is one of the oldest diseases known to man, relatively little is known about the pathogenesis of disease caused by B. mallei. This phenomenon is primarily due to the lack of disease in North America along with the fact that B. mallei can be a particularly dangerous organism to study even in a controlled laboratory environment. Both bacteria are considered BL3 select agents by the CDC.[unreadable] [unreadable] [unreadable] Burkholderia - macrophage interactions:[unreadable] [unreadable] The study of pathogen host cell interactions in vitro is an important tool to define and characterize virulence factors of intracellular bacterial pathogens. The major species of Burkholderia include B. pseudomallei; B. mallei and an avirulent environmentally stable isolate B. thailandensis. B. pseudomallei macrophage interactions have been extensively studied but there is little known about the interactions of B. mallei with macrophages. We have performed a comparative analysis of B. mallei and B. pseudomallei macrophage interactions using the murine macrophage cell line (RAW 264.7). Our findings show that although B. mallei is capable of invading and replicating in RAW cells it is less efficiently internalized and grows more slowly. The optimal multiplicity of infection is critical for permissive B. mallei intracellular growth. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that MOIs significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. We further tested differences in intracellular survival and multiplication among wild type and various mutants of B. mallei and B. pseudomallei. Eighteen mutants produced in each background of B. mallei and B. pseudomallei were tested in the RAW cell infection model. A type III secretion mutant of B. pseudomallei (strain 26bT3) showed marked differences in internalization and growth in RAW cells. An identical B. mallei type III secretion mutant (BMT3) and a B. mallei LPS mutant (GMrmlD) were incapable of growth in RAW cells. The results indicated that in vitro modeling of virulence using RAW macrophages is a simple and credible approach to screen Burkholderia mutants as a rational for analyses in animals.[unreadable] [unreadable] B. mallei is a facultative intracellular pathogen that can cause fatal disease in animals and[unreadable] humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of less than 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-a, IL-6, IL-10, GM-CSF, RANTES and IFN-b when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the[unreadable] outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells.